Haut Zielorgan allergischer Reaktionen auf kleinmolekulare

Substanzen

Hans F Merk

RWTH Aachen University

hansmerkpostrwth-aachende

1968 Beginn der KoKo

bull Praumlsident USA Lyndon B Johnson

bull Bundespraumlsident Heinrich Luumlbke ndash Gustav Heinemann

bull Bundeskanzler Kurt Georg Kiesinger

bull Nobelpreis 1977 Robert W Holley Isolierung einer t-RNA Marshall W Nirenberg PolyU-Experiment Har G Khorana gelang 1970 als erstem die kuumlnstliche Synthese eines Gens

Asthma (IgE) durch kleinmolekulare Substanzen nur nach Sensibilierung (mit Ekzemreaktion) uumlber die Haut

J Pauluhn Toxicology 319 (2014) 10 -22

Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria

recipient of the award in 2003)

Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical

backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959

Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8

Asthma (IgE) durch kleinmolekulare Substanzen nur nach Sensibilierung (mit Ekzemreaktion) uumlber die Haut

J Pauluhn Toxicology 319 (2014) 10 -22

Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria

recipient of the award in 2003)

Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical

backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959

Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8

Stephen Rothman Medal (courtesy of Georg Stingl MD Vienna Austria

recipient of the award in 2003)

Stephen Rothman and the 60th anniversary of the publication of his epic textbook The Physiology and Biochemistry of the Skin In this review we document our belief that Rothman had a seismic impact on moving investigative dermatology from a medical

backwater to a scientific discipline that can hold its own with any other specialty WHC Burgdorf amp DR Bickers JID 2015 Apr135(4)954-959

Steigleder GK Personal experiences with Stephen Rothman MD J Am Acad Dermatol 1988 Sep19(3)596-8

1966 Human repeated insult patch test Albert Kligman

J invest Dermatol 47 (1966) 393 - 409

1966 Human repeated insult patch test Albert Kligman

1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip

1968 Beginn der KoKo

1968 1969

First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin

The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin

DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124

RWTH Aachen1995

Cytochrom P450 Haut

JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

1966 Human repeated insult patch test Albert Kligman

1 Sitzungsprotokoll KoKo 1968 hellipjede absichtliche Sensibilisierung eine Koumlrperverletzunghellip

1968 Beginn der KoKo

1968 1969

First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin

The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin

DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124

RWTH Aachen1995

Cytochrom P450 Haut

JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

1968 Beginn der KoKo

1968 1969

First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin

The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin

DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124

RWTH Aachen1995

Cytochrom P450 Haut

JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

First Evidence Showing that Langerhans Cells Are Involved in Contact Allergic Reactions in Human Skin

The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin

DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124

RWTH Aachen1995

Cytochrom P450 Haut

JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

The skin as a metabolizing organ of pro-haptens Understanding of the fate of reactive chemicals in the skin

DH Kaplan et al Nat Rev Immunol 12 (2012) 114-124

RWTH Aachen1995

Cytochrom P450 Haut

JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

RWTH Aachen1995

Cytochrom P450 Haut

JID 62 (1977) 5-9 JCI 62 (1978) 1061-1068

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

The skin as a metabolizing organ of

pro-haptens

Cytochrom P450

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Activation of prohaptens is mediated by CYPs expressed in skin cells

CYP amount

(pmol)

Skin specific

rhCYP cocktail

CYP1A1 36

CYP1B1 20

CYP2B6 0035

CYP2E1 11

CYP3A5 56

total CYP amount 22

Stratum corneum epidermis

PROHAPTEN

Exte

rn

a

HAPTEN ANTIGEN

PROTEIN ENZYMATIC ACTIVATION

CYP

CYP

content

(pmol)

Liver-like

rhCYP cocktail

CYP1A2 20

CYP2C9 17

CYP2C19 80

CYP2D6 30

CYP2E1 11

CYP3A4 45

Total CYP content 104

(s = slope n = number of PCR cycles x = amount of CYP (pmol)) [Haumlrtter et al 2002] 12

12

101

2 ss

nn

X

X

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TAP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

CYP-cocktail 22pmol 500microl CYP-cocktail 110 pmol 500microl

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Autooxidation and CYP-metabolism important in sensitization to geraniol

Hagvall L et al TaP 233 (2008) 308 ndash 313 CD 67 (2012) 20-27

Neral

Geranial

67-Epoxygeraniol

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Skin sensitization and Regulation

- EU cosmetic directive (EC 12232009) - Registration Evaluation Authorization and Restriction of Chemical Substances REACH (EC 1907 2006)

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin Sensitizers

ARE Antioxidant response element Keap1 Kelch-like ECHndashassociated protein 1 NRF2 nuclear factor-erythroid 2-related factor 2

A Natsch Toxicological Sciences Volume 113 2010 284ndash292

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

KeratinoSensreg-Assay

Luciferase Gene SV40 ARE

Keap1

Nrf2

Hapten

Luminescence Luciferase Gene SV40 ARE

Keap1

Nrf2

Nrf2

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

2 days

30min

KeratinoSens-Cells

Viability

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Modified KeratinoSensreg Assay

Hapten Pro-

hapten

Luminescence

Pro-hapten

S9

CYP Cocktail CYP1A1

CYP1B1

CYP2B6

CYP2E1

CYP3A5

2 days

30min

KeratinoSens-Cells

Viability

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

KeratinoSens with Carvoxime

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Eugenol - Creosol

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

KeratinoSens mit Creosol

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Methods 2

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

HSQC (2D) spectra of treated samples epidermal layers (left) and dermis (right)

Creo

Creo

Creo

12 benzoquinone-hapten-protein-adduct

hapten-protein-adduct

Methandiol (monohydrated formaldehyd)

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Bioactivationreactivity of creosol-13C2 in 3D skin equivalents

Epidermis + Dermis

Dermis

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Haut Zielorgan allergischer Reaktionen auf kleinmolekulare Substanzen

Dalbergia latifolia (Palisander) R-4-Methoxydalbergion 1

S-4-Methoxydalbergion 1

Obtusachinon 1

S-44-Dimethoxydalbergion 1

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion

Haut Zielorgan allergischer Reaktionen auf

kleinmolekulare Substanzen

0

5

10

15

20

25

(S)-44DMD [R]-4-MD (S)-4-MD

10(-8)M

5x10(-8)M

10(-7)M

5x10(-7)M

10(-6)M

5x10(-6)

10(-5)M

SI= Stimulation index lt2 no increased proliferation 2-3 prolif questionable gt3 increased proliferation

SI

Klinik fuumlr Dermatologie amp Allergologie

PHA 1320 Tetanustoxin 575 TT+solvent 585 Solvent 108

M

DMD Dimethoxydalbergion MD Methoxydalbergion